Second-harmonic imaging microscopy of normal human and keratoconus cornea

PURPOSE: The purpose of this study was to evaluate the ability of second-harmonic imaging to identify differences in corneal stromal collagen organization between normal human and keratoconus corneas. METHODS: Six normal corneas from eye bank donors and 13 corneas of patients with keratoconus, obtained after penetrating keratoplasty were examined. A femtosecond titanium-sapphire laser with 800-nm output was used to generate second-harmonic signals collected at 400 nm from central and paracentral corneal tissue blocks. Three-dimensional (3-D) data sets were collected and reconstructed to evaluate the location and orientation of stromal collagen lamellae. RESULTS: Imaging of second-harmonic signals combined with 3-D reconstruction of the normal cornea identified a high degree of lamellar interweaving, particularly in the anterior cornea. Of note was the detection of lamellae that inserted into Bowman's layer and were oriented transverse to the corneal surface, penetrating posteriorly approximately 120 mum. In keratoconus corneas, imaging second-harmonic signals identified less lamellar interweaving and a marked reduction or loss of lamellae inserting into Bowman's layer in 12 of 13 cases, particularly in regions associated with cone development without breaks in Bowman's layer or scarring. CONCLUSIONS: Compared with normal adult corneas, marked abnormalities were detected in the organization of the anterior corneal collagen lamellae of keratoconus corneas by second harmonic imaging. These structural abnormalities are consistent with the known changes in collagen organization and biomechanical strength of keratoconus.     

Confocal microscopy of the human cornea in vivo

In vivo, scanning-slit, confocal microscopy offers improved resolution and has resulted in new discoveries of corneal pathology at the cellular level.The ability to provide high resolution, real-time images of the full thickness of theliving human cornea gives the clinician and the researcher an important new tool.     

In vivo confocal microscopy of the human cornea

AIMS: To describe the optics of in vivo confocal microscopy, its advantages over previous methods, and to summarise the literature that arose from its use for the observation of the human cornea. A critical review of the clinical usefulness of this new technology for the corneal examination is undertaken. METHODS: Confocal microscopes obtain increased resolution by limiting the illumination and observation systems to a single point. Rapid scanning is used to reconstruct a full field of view and allows for "real time" viewing. RESULTS: Coronal sections of the in situ epithelium, Bowman's membrane, stroma, and endothelium can be visualised at a resolution of 1-2 micro m. A backscattered light intensity curve allows objective measurements of sublayer thickness and corneal haze to be taken. In vivo confocal microscopy is therefore particularly useful in the areas of infective keratitis, corneal dystrophies, refractive surgery, and contact lens wear, where it aids in differential diagnosis and detection of subtle short and long term changes. Real time endothelial cell assessment can also be performed. CONCLUSION: Because of their ability to visualise living tissue at cellular levels, confocal microscopes have proved useful additions to the current clinical tools.     

Scanning electron microscopy of normal human corneal epithelium obtained by scraping-off in vivo

The surface of normal human corneal epithelium was examined by scanning electron microscopy (SEM). The study was carried out on specimens obtained by scraping-off in vivo, and immediately fixed. Four different types of microprojections were observed on the epithelial surface: microridges, microvilli, tufted microvilli and knobs. Detailed high magnification of surface microprojections showed that they consist of subunits. Our data suggest a correlation between surface morphology and maturity of the corneal epithelial cell. An evolutive sequence is proposed, from the youngest cell, provided with microridges, to the oldest exfoliating one, with knobs.     

Epithelial dendritic cell distribution in normal and inflamed human cornea: in vivo confocal microscopy study

PURPOSE: To evaluate dendritic cell (DCs) density, distribution, and morphology in central corneal and limbal epithelium in normal subjects and patients with immune-mediated corneal inflammation using in vivo confocal microscopy (IVCM). DESIGN: Comparative case-controlled, observational confocal microscopy study. METHODS: A total of 135 eyes of 135 patients were investigated. Group 1 (normal eyes) included 45 eyes of 45 healthy volunteers, group 2 photorefractive keratectomy (PRK-treated eyes) included 45 myopic eyes of 45 patients treated with PRK, and group 3 (inflamed eyes) comprised 45 eyes of 45 patients affected by immune-mediated corneal inflammation. The central cornea and limbus were examined for epithelial dendritic-shaped cells using laser scanning IVCM. DCs density was calculated using image analysis software. RESULTS: Cells with a branching dendritic morphology were visualized in the basal epithelial layer, basal lamina, and subbasal nerve plexus, in the central cornea, and in the basal layer and basal membrane of the limbal epithelium. The limbal epithelium demonstrated DCs in 93.3%, 89%, and 97.7% of eyes in group 1, 2, and 3, respectively (P = ns). Central epithelial DCs were observed in 20.0% and 13.3% of eyes in group 1 and 2 (P = ns), while in 93.3% of eyes in group 3 (P < .001). DCs were found to be significantly higher at the limbus compared with central cornea in each group (P < .001). Cell densities observed in group 3 were significantly greater than groups 1 and 2, at both locations (P < .05). CONCLUSIONS: Laser scanning IVCM is a useful method for evaluating epithelial DCs distribution at the limbus and central cornea in both healthy and diseased eyes.     

Age-related differences in the normal human cornea: a laser scanning in vivo confocal microscopy study

AIMS: To quantify and establish baseline normative data for age-related differences in cellular and innervation density in the normal, healthy, human cornea using laser scanning in vivo confocal microscopy. METHODS: Cross-sectional study of 85 normal subjects assessed via corneal topography and laser scanning in vivo confocal microscopy. RESULTS: Mean age was 38+/-16 years (range 18-87 years) and 60% of subjects were female. Anterior keratocyte density declined by 0.9% per year (r = -0.423, p<0.001), posterior keratocyte density declined by 0.3% per year (r = -0.250, p = 0.021) and endothelial cell density declined by 0.5% per year (r = -0.615, p<0.001). Sub-basal nerve fibre density declined by 0.9% per year (r = -0.423, p<0.001). No association was observed between age and basal epithelial cell density, or between age and central corneal thickness, corneal astigmatism or horizontal corneal diameter (p>0.05). No association was observed between subject gender and corneal cell or innervation density. CONCLUSIONS: Using laser scanning in vivo confocal microscopy this study highlights a significant, and relatively linear, reduction in keratocyte and endothelial cell density with increasing subject age. Interestingly, corneal sub-basal nerve fibre density also significantly decreases with increasing age. In vivo laser scanning confocal microscopy provides a safe, non-invasive method for the establishment of normative data and assessment of alterations in human corneal microstructure following surgery or disease processes.     

Scanning electron microscopy of cultures of macular dystrophy of the cornea]

The scanning electron microscope shows that the keratocytes of the macular dystrophy of the cornea have an irregular and anfractuous surface and that some cytoplasmic particles are broken off. This explains the isolated elements found in the stroma.     

Three-dimensional confocal microscopy of the human cornea in vivo

This paper reviews the literature from 1990 to 2000 and evaluates the seminal investigations performed with the confocal microscope on the in vivo human cornea. Our pedagogical technique is to illustrate both the advantages and the problems associated with occular confocal microscopy by way of annotated examples. Confocal microscopy offers improved resolution and has resulted in new discoveries of corneal pathology at the cellular level. The ability to provide high-resolution, real-time images of the full thickness of the cornea gives the clinician and the researcher an important new tool for the investigation of the cornea.     

T lymphocytes of the normal human cornea.

Lymphocytes in the periphery of the normal human cornea are identified as being only T lymphocytes by immunohistochemical methods. OKT-4 positive cells (T-helper/inducer lymphocytes) and OKT-8 positive cells (T-suppressor/cytotoxic lymphocytes) are found in similar numbers in most of the corneas examined. OKT-4 positive cells in the cornea present a risk of transferring HTLV-III (HIV) by corneal grafting.     

Scanning electron microscopy of human drusen

Drusen are small, yellowish deposits that form under the retinal pigment epithelium (RPE) with senescence or under certain pathological conditions. The present study examined these structures under the scanning electron microscope. Tissue came from four eyes of 66- and 75-year-old donors who demonstrated widespread drusen of the posterior fundus noted on postmortem examination. Specimens were prepared by either detaching the RPE from Bruch's membrane, or by cryofracturing the tissue for cross-sectional views. Drusen appeared to be composed of irregularly-shaped globular masses and of distinct spherical entities. These particles varied greatly in size, and were situated between the RPE's basement membrane and the outer collagenous zone of Bruch's membrane. Surface views showed drusen components to be embedded in the collagenous zone of Bruch's membrane. Pits corresponding to the sizes of the globular and spherical masses imply that some particles were lost during tissue processing. Fractured cross sections of the irregularly-shaped globular masses revealed a homogeneous, granular matrix with no distinct ultrastructural features, while some of the fractured spherical components demonstrated an internal core. Transmission electron microscopic analysis on the same specimens that were subjected to SEM corroborated these observations. Analytical x-ray microanalysis (Kevex, Foster City, CA) in the SEM revealed major peaks for calcium and phosphorous in the crystalline spherical components, and primarily potassium and chloride in the globular structures.     

In vivo confocal microscopy of human cornea covered with human amniotic membrane

Purpose  Amniotic membrane transplantation has been widely performed to reconstruct the surface of the eye and treat chemical burns or epithelial defects. However, we have difficulty observing the cornea through the opaque transplanted amniotic membrane by slit-lamp biomicroscopy. We investigated the use of confocal microscopy for observation of human corneas covered with amniotic membrane. Methods  Human amniotic membrane was placed onto the normal corneas of five volunteers aged 22–24 years. Then, all layers of the covered corneas were observed by in vivo confocal microscopy. Results  Confocal microscopy displayed the epithelium, basement membrane, and stroma of the amniotic membrane. It also displayed the corneal epithelium. Furthermore, corneal stromal keratocytes and the corneal endothelium were clearly observed through the amniotic membrane by confocal microscopy. Conclusions  We demonstrated that in vivo confocal microscopy enabled us to observe all layers of corneas covered with amniotic membrane in normal human eyes. Our findings suggest that confocal microscopy may have advantages for clinical examination of the ocular surface, including all layers of the cornea.     

Histology and transmission electron microscopy of the cornea in xeroderma pigmentosum type C

Xeroderma pigmentosum is a very rare precancerous skin disease that is triggered by sunlight. It is caused by a defect in the DNA repair system and causes benign and malignant transformations. Only eye tissues that come into contact with UV light are affected, such as the lids, conjunctiva and cornea. We describe a patient who suffered from xeroderma pigmentosum type C, showing the typical skin alterations but no sign of malignancy. A perforating keratoplasty was performed on both eyes because of the dense opacity of the corneas. The corneal buttons obtained were examined by light and transmission electron microscopy. Degeneration was found only in the basal-cell layer of the corneal epithelium. The most severe morphological changes were seen in Bowman's layer, the subepithelial stroma, Descemet's membrane and the corneal epithelium. Bowman's layer was often interrupted or replaced by a degenerative pannus, which extended into the underlaying stroma. Subepithelial channels were localized in the basal epithelium and protruded into the subepithelial stroma. In both corneas, Descemet's membrane contained different amounts of so-called lattice collagen, and the remaining endothelial cells in the left cornea contained numerous melanin granules. Offprint requests to: E.-M. Haller     

Localization of acetylcholinesterase in the rabbit cornea by light and electron microscopy.

Acetylcholinesterase (AChE) has been localized in the rabbit cornea by light and electron microscopy histochemical techniques. In the stroma, the enzyme is concentrated in nerves. In the epithelium, the enzyme is concentrated in intercellular spaces devoid of nerves. The morphologic appearance of the enzyme staining by light and electron microscopy in the epithelium is similar; consequently, the staining demonstrated with light microscopy examination does not always represent epithelial nerves. A significant portion of corneal acetylcholinesterase therefore appears unrelated to nerves. Considerably smaller deposits of enzyme reaction product were present in cells in every layer of the cornea, using electron microscopy histochemistry; they were not identified by light microscopy.     

Surface ultrastructure of collagen fibrils and their association with proteoglycans in human cornea and sclera by atomic force microscopy and energy-filtering transmission electron microscopy

PURPOSE: We aimed to investigate the possible association of proteoglycans with D-periodic collagen fibrils in the human cornea and sclera, using energy-filtering transmission electron microscopy (EF-TEM) and atomic force microscopy (AFM). METHODS: Human cornea and sclera were digested with keratanase to eliminate keratan sulfate proteoglycans (KSPGs). For EF-TEM observation, surface proteoglycans were detected by cupromeronic blue (CB) staining. For AFM observation, cornea and sclera were treated with sodium hydroxide before and after keratanase digestion, and the surface topology of collagen fibrils was analyzed. RESULTS: With CB staining, numerous CB-positive short filaments of surface proteoglycans (proteoglycan filaments) were observed in the interfibrillar spaces of cornea and sclera associated with collagen fibrils. AFM imaging showed that the depth and periodicity of D-periodic collagen fibrils in keratanase-treated corneal collagens were deeper and more regular than in untreated ones. Moreover, the depth and periodicity of keratanase-untreated corneal collagens were shallow and irregular in comparison with keratanase-untreated scleral collagens. On the other hand, there was no difference in depth or regularity between keratanase-treated and -untreated scleral collagen fibrils. Using AFM imaging, additional thin grooves sub-bands were detected on the surface of keratanase-treated corneal collagen fibrils. The grooves were not detected in keratanase-untreated collagen fibrils nor in scleral collagen fibrils with or without keratanase digestion. Comparing densitometry waves, the grooves of D-periodic corneal collagen sub-bands corresponded to a and c bands. CONCLUSION: Using AFM and EF-TEM to study corneal and scleral collagen fibrils and their association with proteoglycans, we conclude that KSPG is found in ample amounts in the human cornea in comparison with sclera. Moreover, we topologically detected KSPG attached to a and c bands of collagen fibrils.     

Confocal microscopy of the cornea. Communication 1. The normal morphological pattern

The paper presents the results of life-time confocal microscopy using a Confoscan-4 confocal microscope (Nidek). The study was conducted on 10 healthy volunteers (20 eyes), which evaluated the specific features of the normal morphological pattern of the cornea. The morphological features of three types of anterior corneal epithelial (basal, wing, and spiny) cells were revealed. The possibilities of visualizing subbasal and stromal nerve fibers were assessed. Corneal stromal layers (these being anterior, anteromedian, median, and posterior) differing in the density of cellular elements, their shape, and orientation) were conventionally identified. In health, Bowman's and Descemet's membranes were not visualized as they were transparent and failed to reflect light. The posterior corneal epithelium was microscopically presented by a layer of light hexagonal or polygonal squamous cells with well-defined outlines. Thus, life-time corneal confocal microscopy is a highly informative noninvasive study.     

不同光源共焦显微镜的活体正常角膜图像研究

目的 分别采用卤素灯光源共焦显微镜和激光光源共焦显微镜对中国正常成人活体角膜各层组织结构进行观察,对照研究其图像特征.方法 对35例(70只眼)中国成人(年龄18～55岁)中央部角膜分别采用卤素灯光源共焦显微镜和激光光源共焦显微镜进行检查,研究角膜各层结构的图像特点,并进行对比.结果 共焦显微镜检查的35例(70只眼)中,67只眼95.7％成功获得了角膜上皮翼状细胞层的图像,70只眼100％成功获得角膜上皮基底细胞层、前弹力层、前基质层、后基质层及角膜内皮细胞层的图像.结论 卤素灯光源共焦显微镜的放大倍数是1000倍,细胞放大倍数大,近似于初级电镜的放大倍数,但卤素灯光源的组织穿透力较弱,光源易衰减,所得的角膜各层细胞图像较激光角膜共焦显微镜的略微模糊,所得角膜各层图像总体颜色偏灰、结构偏模糊.激光角膜共焦显微镜的放大倍数是800倍,较卤素灯光源的共焦显微镜略小,但激光光源的组织穿透力强,所得角膜各层细胞图像较卤素灯光源的更清晰,而且在特定角度可获得非常类似角膜组织病理学切片的图像.     

Lymphocytes and Langerhans cells in the normal human cornea

The distribution of B- and T-lymphocytes, of OKIa positive cells, and of HLA-ABC antigens in the normal cornea was investigated using monoclonal antibodies. The lymphocytes and Langerhans cells are present mainly in the well-vascularized limbic region but also occur albeit in small number in the center of the cornea. HLA-ABC antigens are strongly expressed on the epithelial cells of the cornea and the limbus.